Identification and purification of a functional amine transporter from bovine chromaffin granules.
The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isoforms with different pI values have been separated and shown to be active. One with an unusually acidic pI (approximately 3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 microM and a Vmax of 140 nmol/mg/min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.
Stern-Bach
Y
Y
Department of Molecular Biology, Hebrew University Hadassah Medical School, Ein Karem, Jerusalem, Israel.
Greenberg-Ofrath
N
N
Flechner
I
I
Schuldiner
S
S
D000818:Animals; D001679:Biogenic Amines / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003902:Detergents; D007425:Intracellular Membranes / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011510:Proteolipids / Q000378:metabolism; D012110:Reserpine / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism*
D001679:Biogenic Amines; D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; D008562:Membrane Glycoproteins; D011510:Proteolipids; C020485:proteoliposomes; D012701:Serotonin; D012110:Reserpine